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1330
VOLUME 113 |NUMBER 10 | October 2005
•
Environmental Health Perspectives
Research
Arsenic is a ubiquitous element distributed in
the environment, and millions of people are
chronically exposed to arsenic worldwide
(Abernathy et al. 1999). Naturally contami-
nated drinking water is the main source of
arsenic exposure, posing potential risk to
human health (Nordstrom 2002; Schoen et al.
2004; Smith et al. 2002). Chronic arsenic
exposure has been associated with a wide range
of illnesses including cancer, hyperkeratosis,
diabetes, and cardiovascular disease (Engel
et al. 1994; Rossman 2003; Tseng 2004; Yu
et al. 1984). Cardiovascular effects of arsenic
exposure include hypertension, atherosclerosis,
cerebrovascular disease, ischemic heart disease,
and peripheral vascular disorders such as black-
foot disease (resulting from gangrene caused by
obstruction of peripheral blood vessels) in
humans (Chen et al. 1995, 1996; Chiou et al.
1997; Rahman et al. 1999; Simeonova et al.
2003; Wang et al. 2002).
Lee et al. (2002) recently suggested that
the mechanism for arsenic-induced cardio-
vascular disease is the increased susceptibility
of platelets to aggregate, resulting in enhanced
arterial thrombosis. Other mechanisms may
also be responsible for the diversity of human
cardiovascular disease from chronic arsenic
exposure. One possibility is that arsenic may
alter the normal vasomotor tone of blood
vessels, which rises from contractility of vas-
cular smooth muscle cells.
The contraction of smooth muscle is regu-
lated by mediators such as neural and humoral
factors, mechanical forces, and vasoactive sub-
stances from endothelial cells. Vascular smooth
muscle contraction is triggered primarily by a
rise in intracellular free Ca
2+
concentration
(Sanders 2001). Ca
2+
binds to calmodulin
(CaM), allowing Ca
2+
–CaM complex forma-
tion, which binds to and activates myosin light
chain kinase (MLCK) (Horowitz et al. 1996).
The active MLCK catalyzes the phosphoryla-
tion of the regulatory myosin light chain
(MLC), which then triggers myosin–actin
interaction, leading to the shortening of mus-
cle and generation of force. When the intra-
cellular Ca
2+
concentration returns to a lower
level, myosin is dephosphorylated by myosin
phosphatase and the muscle relaxes (Somlyo
and Somlyo 1994; Walsh et al. 1994).
In addition to the contraction mediated
by Ca
2+
-dependent MLCK, it has been
recently suggested that smooth muscle con-
traction is modulated by Ca
2+
sensitization
(Somlyo and Somlyo 2000). This implies that
Ca
2+
-dependent contractions occur, but at a
lower Ca
2+
concentration than would be
expected for that mediated directly via MLCK
(Somlyo and Somlyo 2000). An increase in
MLC phosphorylation due to reduced activity
of myosin phosphatase appears to result in
the hypercontraction of blood vessels, which
is a characteristic feature of Ca
2+
sensitization
(Fukata et al. 2001; Uehata et al. 1997). This
abnormal hypercontraction is generally
known to cause acute vasospasm, micro-
circulatory ischemia, increased systemic blood
pressure, and ultimately, possible vascular dis-
eases (Bohr et al. 1991; Mohri et al. 1998;
Mulvany and Aalkjaer 1990).
Previous epidemiologic studies have
reported that peripheral vascular resistance and
systemic blood pressure were elevated in popu-
lations that had ingested the arsenic-contami-
nated drinking water (Chen and Yen 1964;
Tseng et al. 1995). Carmignani et al. (1985)
observed that chronic administration of arsen-
ite to rats and rabbits caused a significant
increase in vascular peripheral resistance. These
studies suggest the possibility that arsenic may
disrupt normal vasomotor function, leading to
hypercontraction of blood vessels. Indeed, our
previous study demonstrated that arsenic
could inhibit acetylcholine-induced vascular
relaxation via inhibition of nitric oxide syn-
thase in vascular endothelial cells (Lee et al.
2003). In the process of investigating the
effect of arsenic on blood vessels, we observed
that arsenic could enhance agonist-induced
contraction in an aortic ring organ bath sys-
tem, suggesting that arsenic could disrupt
contractile function in vascular smooth mus-
cles as well. Therefore, in the present study we
investigated the mechanism of arsenic-
induced vascular dysfunction and its possible
contribution to cardiovascular diseases.
Materials and Methods
Chemicals. The following chemicals were pur-
chased from Sigma (St. Louis, MO, USA):
sodium arsenite (trivalent inorganic arsenic),
Address correspondence to J.-H. Chung, College of
Pharmacy, Seoul National University, Shinrim-dong
San 56-1, Seoul 151-742, Korea. Telephone: 82-2-
880-7856. Fax: 82-2-885-4157. E-mail: jhc302@
plaza.snu.ac.kr
This work was supported by the Ministry of
Science and Technology National Research and
Development Program and by the Eco-Technopia
21 project of the Ministry of Environment, Korea.
The authors declare they have no competing
financial interests.
Received 7 February 2005; accepted 14 June 2005.
Inorganic Arsenite Potentiates Vasoconstriction through Calcium
Sensitization in Vascular Smooth Muscle
Moo-Yeol Lee,1Young-Ho Lee,2Kyung-Min Lim,1Seung-Min Chung,1Ok-Nam Bae,1Heon Kim,3
Choong-Ryeol Lee,4Jung-Duck Park,5and Jin-Ho Chung1
1College of Pharmacy, Seoul National University, Seoul, Korea; 2College of Medicine and BK21 Project for Medical Sciences, Yonsei
University, Seoul, Korea; 3College of Medicine, Chungbuk National University, Cheongju, Korea; 4Ulsan University Hospital, Ulsan,
Korea; 5College of Medicine, Chung-Ang University, Seoul, Korea
Chronic exposure to arsenic is well known as the cause of cardiovascular diseases such as hyper-
tension. To investigate the effect of arsenic on blood vessels, we examined whether arsenic affected
the contraction of aortic rings in an isolated organ bath system. Treatment with arsenite, a tri-
valent inorganic species, increased vasoconstriction induced by phenylephrine or serotonin in a
concentration-dependent manner. Among the arsenic species tested—arsenite, pentavalent inor-
ganic species (arsenate), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV)—
arsenite was the most potent. Similar effects were also observed in aortic rings without
endothelium, suggesting that vascular smooth muscle plays a key role in enhancing vaso-
constriction induced by arsenite. This hypercontraction by arsenite was well correlated with the
extent of myosin light chain (MLC) phosphorylation stimulated by phenylephrine. Direct Ca2+
measurement using fura-2 dye in aortic strips revealed that arsenite enhanced vasoconstriction
induced by high K+without concomitant increase in intracellular Ca2+ elevation, suggesting that,
rather than direct Ca2+ elevation, Ca2+ sensitization may be a major contributor to the enhanced
vasoconstriction by arsenite. Consistent with these in vitro results, 2-hr pretreatment of 1.0 mg/kg
intravenous arsenite augmented phenylephrine-induced blood pressure increase in conscious rats.
All these results suggest that arsenite increases agonist-induced vasoconstriction mediated by MLC
phosphorylation in smooth muscles and that calcium sensitization is one of the key mechanisms
for the hypercontraction induced by arsenite in blood vessels. Key words: arsenic, arsenite, blood
vessels, calcium sensitization, cardiovascular disease, myosin light chain phosphorylation, vaso-
constriction. Environ Health Perspect 113:1330–1335 (2005). doi:10.1289/ehp.8000 available via
http://dx.doi.org/ [Online 14 June 2005]
sodium arsenate (pentavalent inorganic
arsenic), dimethylarsinic acid (DMA
V
),
phenylephrine, and serotonin creatinine sulfate.
We obtained monomethylarsonic acid
(MMA
V
) from Chem Service (West Chester,
PA, USA) and anti-MLC and anti-phospho-
MLC antibody from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Fura-2/AM was sup-
plied by Molecular Probe (Eugene, OR, USA),
and all other reagents used were of the highest
purity available.
Animals. The entire animal protocol was
approved by the Ethics Committee of Animal
Service Center at Seoul National University.
We used male Sprague-Dawley rats (Dae
Han BioLink Co., Seoul, Korea) weighing
300–400 g throughout all experiments. Before
the experiments, animals were acclimated for
1 week in the laboratory animal facility main-
tained at constant temperature and humidity
with a 12-hr light/dark cycle. Food and water
were provided ad libitum.
Measurement of vasoconstriction in
organ bath. Rats were sacrificed by decapita-
tion and then exsanguinated. We carefully
isolated the thoracic aorta and cut it into ring
segments. Aortic rings without endothelium
were prepared by gently rubbing the intimal
surface of the aortic rings with a wooden
stick. We then mounted the rings in four-
channel organ baths filled with Krebs-Ringer
(KR) solution (115.5 mM NaCl, 4.6 mM
KCl, 1.2 mM KH
2
PO
4
, 1.2 mM MgSO
4
,
2.5 mM CaCl
2
, 25.0 mM NaHCO
3
, and
11.1 mM glucose, pH 7.4). The organ bath
was continuously gassed with 95% O
2
/5%
CO
2
and maintained at 37°C. The rings
were stretched gradually to an optimal resting
tension of 2 g and equilibrated for 30 min.
The change in tension was measured isomet-
rically with Grass FT03 force transducers
(Grass Instrument Co., Quincy, MA, USA)
and recorded using the AcqKnowledge III
computer program (BIOPAC Systems Inc.,
Goleta, CA, USA).
To investigate the effect of arsenic on vaso-
constriction, we treated the aortic rings with
arsenic or the vehicle (saline) in minimum
essential media (MEM) with 100 U/mL peni-
cillin and 100 µg/mL streptomycin in a 95%
air/5% CO
2
incubator for 14 hr at 37°C. After
mounting the aortic rings pretreated with
arsenic in organ baths, we induced the contrac-
tion by cumulatively adding phenylephrine or
serotonin to obtain concentration-contraction
curves. In the experiments of high K
+
-induced
contraction, 100 mM KCl-containing KR
solution prepared by substituting K
+
with Na
+
was cumulatively added to the bath to obtain
the indicated final concentrations.
Measurement of MLC phosphorylation.
We measured the extent of MLC phosphory-
lation using polyacrylamide gel electrophore-
sis (PAGE) and immunoblot analysis using an
anti-phospho-MLC antibody, as previously
described (Sakurada et al. 1998). After the
aortic rings were pretreated with various con-
centrations of arsenite for 14 hr, 10
-8
M
phenylephrine was added to the organ bath
for 2 min, and then ice-cold acetone with
10% trichloroacetic acid and 10 mM dithio-
threitol was immediately added to stop the
reaction. The aortic rings were washed with
the acetone solution three times, lyophilized
overnight, and stored at –70°C until protein
extraction. After the dried tissues were cut
into small pieces, we extracted proteins in a
50 µL sample buffer containing 8 M urea,
2% sodium dodecyl sulfate, 5% β-mercapto-
ethanol, 0.01% bromophenol blue, and
62.5 mM Tris-HCl by vortexing for 3 hr at
room temperature. Protein extracts were
electrophoresed in a 15% polyacrylamide
mini-slab gel (Bio-Rad, Hercules, CA, USA)
and then transferred onto nitrocellulose mem-
branes in Tris/glycine buffer (25 mM Tris,
192 mM glycine). We detected the MLC
level and the extent of phosphorylation
with immunoblotting using anti-MLC anti-
body and anti-phospho-MLC antibody
(Santa Cruz Biotechnology), respectively.
Immunoreactive bands were visualized by
horseradish peroxidase-conjugated secondary
antibody (Santa Cruz Biotechnology) and an
enhanced chemiluminescence kit (ECL;
Amersham, Buckinghamshire, UK). For den-
sitometric analysis of MLC-P/MLC, we deter-
mined densities of the corresponding bands
using TINA software (Raytest, Straubenhardt,
Germany).
Measurement of intracellular calcium level.
To measure intracellular Ca
2+
levels, we cut the
thoracic aorta without endothelium into spiral
strips approximately 8 mm in length and
1 mm in width under a dissecting microscope.
After the aortic strips were treated with arsen-
ite for 14 hr, they were exposed to 10 µM
acetoxymethyl ester of fura-2 (fura-2/AM) and
0.1% cremophor EL in a Krebs-Henseleit
solution [(KH) solution: 119 mM NaCl,
4.6 mM KCl, 1.2 mM KH
2
PO
4
, 1.5 mM
MgSO
4
, 2.5 mM CaCl
2
, 25.0 mM NaHCO
3
,
and 11 mM glucose, pH 7.4] for 4 hr at room
temperature.
We measured the intracellular free Ca
2+
level based on the method described by Ozaki
et al. (1991). Fure-2-loaded muscle strips were
held horizontally in the organ chamber of a
fluorimeter (CAF-110; Jasco, Tokyo, Japan)
filled with KH solution. One end of the mus-
cle strip was connected to a force-displacement
transducer to monitor vessel tones. The KH
solution was maintained at 37°C and continu-
ously aerated with 95% O
2
/5% CO
2
. Passive
tension of 2 g was applied and allowed to equi-
librate before measurement. We elicited mus-
cle contractions by changing the media with
the KH solution containing 12.5, 25, and
90 mM KCl. Muscle strips were illuminated
alternatively at 48 Hz in excitation wavelengths
of 340 and 380 nm. We measured the inten-
sity of 500 nm fluorescence emitted by
340 nm excitation (F
340
) and that emitted by
380 nm (F
380
) successively. We calculated the
ratio of F
340
to F
380
[R(F
340
/F
380
)] as an indi-
cator of intracellular Ca
2+
.
Measurement of blood pressure change
induced by phenylephrine. Rats were anes-
thetized with phenobarbital (50 mg/kg,
intraperitoneal). We placed a catheter of poly-
ethylene PE-50 tubing (Clay Adams, Sparks,
MD, USA) filled with heparinized saline
(100 U/mL) in the carotid artery to measure
blood pressure, and we placed a catheter of
polyethylene PE-10 fused to PE-50 tubing in
the jugular vein to administer drugs. Catheters
were tunneled subcutaneously and exteriorized
at the back of the neck. Wounds were sutured
and cleaned with alcohol. Experiments were
performed after a 1-day recovery period. On
the day of the experiment, the arterial catheter
was connected to a pressure transducer
(BIOPAC Systems Inc.) and blood pressure
was measured using the AcqKnowledge III
Enhancement of vasoconstriction induced by arsenic
Environmental Health Perspectives
•
VOLUME 113 |NUMBER 10 | October 2005
1331
BA
98765 7 6 5 4
3
2
1
0
4
3
2
1
0
Contraction (g)
Contraction (g)
Log [phenylephrine (M)] Log [serotonin (M)]
Control
Arsenite 5 µM
Arsenite 10 µM
Arsenite 25 µM
*
*
*
*
Figure 1. Enhancement of phenylephrine- and serotonin-induced vasoconstriction in aortic rings by arsenite.
(
A
) Phenylephrine. (
B
) Serotonin. See “Materials and Methods” for details. Values shown are mean ± SE of
four independent experiments.
*Significantly different from the corresponding control (
p
< 0.05).
computer program. We allowed blood pres-
sure to stabilize for a minimum of 30 min
before beginning treatment. To determine the
effects of arsenite on blood pressure increase
induced by phenylephrine, we administered
arsenite solution (1.0 mg/kg) by an intra-
venous bolus injection into the jugular vein.
In the controls, equivalent amounts of saline
were injected. After 2 hr, we infused the rats
with 2.5 µg/kg/min phenylephrine for 3 min
via the jugular vein and monitored the change
of blood pressure in response to phenylephrine
simultaneously. Infusions were performed
with a Harvard syringe pump (South Natick,
MA, USA) at a rate of 0.1 mL/min.
Statistical analysis. We calculated the
means and standard errors for all treatment
groups. The data were subjected to one- or
two-way analysis of variance (ANOVA) fol-
lowed by Duncan’s multiple range test to
determine which means were significantly dif-
ferent from the control. Statistical analysis
was performed using SPSS software (SPSS
Inc., Chicago, Il, USA). In all cases, we used a
p-value of < 0.05 to determine significance.
Results
To investigate whether arsenic affects contrac-
tion of blood vessels, we treated intact aortic
rings with various concentrations of arsenite
(trivalent inorganic arsenic) for 14 hr and then
added phenylephrine and serotonin cumula-
tively to obtain concentration-contraction
curves. Arsenite alone did not cause any
changes in basal vascular tone (data not shown).
Arsenite treatment, however, enhanced the con-
traction induced by both phenylephrine and
serotonin in a concentration-dependent man-
ner (Figure 1). We investigated the effects of
pentavalent inorganic species (arsenate) and two
major metabolites, MMA
V
and DMA
V
, on
phenylephrine-induced constriction (Figure 2).
Arsenate did enhance phenylephrine-induced
vasoconstriction, but the concentration
required was higher than that of arsenite.
MMA
V
and DMA
V
up to 100 µM failed to
affect the phenylephrine-induced vasoconstric-
tion. These results suggest that agonist-
induced contraction in blood vessels can be
enhanced by arsenic and that arsenite is the
most potent form tested.
To investigate whether the enhanced
contraction induced by arsenite was an
endothelium-dependent effect, we performed
experiments using aortic rings without
endothelium. Treatment with arsenite to aor-
tic rings without endothelium still resulted in
a concentration-dependent increment of ago-
nist-induced contraction in blood vessels
(Figure 3), suggesting that the enhanced con-
traction by arsenite is primarily due to hyper-
contraction of smooth muscles. To examine
whether hypercontraction by arsenite is medi-
ated by the phosphorylation of MLC in
smooth muscles, we evaluated the effect of
arsenite on phenylephrine-induced MLC
phosphorylation in aortic rings without
endothelium. Arsenite treatment alone did
not alter the basal levels of MLC phosphory-
lation (Figure 4A). Addition of phenylephrine
alone did not affect the total MLC levels, but
it increased MLC phosphorylation signifi-
cantly (Figure 4B,C). However, when the
aortic rings without endothelium were stimu-
lated with phenylephrine, arsenite treatment
resulted in a significant increase in MLC
phosphorylation without a concomitant
change in MLC levels (Figure 4B,C). These
results indicate that arsenite enhances agonist-
induced vasoconstriction through MLC phos-
phorylation in smooth muscles.
To examine whether the increased MLC
phosphorylation was mediated by intracellular
Ca
2+
elevation in vascular smooth muscles, we
investigated the effects of arsenic on intra-
cellular Ca
2+
levels when vasoconstriction was
initiated by high K
+
concentration. We used a
high concentration of K
+
to induce contrac-
tion in blood vessels, since K
+
directly induces
rapid influx of extracellular Ca
2+
through
voltage-gated Ca
2+
channels in plasma mem-
brane without involving other signal trans-
duction pathway and thus could serve as a
simple alternative tool to investigate the cur-
rent premise. As shown in Figure 5A, 25 µM
arsenite enhanced vasoconstriction induced
Lee et al.
1332
VOLUME 113 |NUMBER 10 | October 2005
•
Environmental Health Perspectives
98765
3
2
1
0
Contraction (g)
Log [phenylephrine (M)]
Control
Arsenate 100 µM
MMA 100 µM
DMA 100 µM*
Figure 2. Effect of arsenic species on contraction
of aortic rings induced by phenylephrine. See
“Materials and Methods” for details. Values shown
are mean ± SE of five independent experiments.
*Significantly different from the control (
p
< 0.05).
3
2
1
0
0 5 10 25 0 5 10 25
Arsenite (µM) Arsenite (µM)
Contraction (g)
Contraction (g)
**
*
*
BA
3
2
1
0
Figure 3. Enhancement of phenylephrine- and serotonin-induced contraction by arsenite in aortic rings
without endothelium. (
A
) Phenylephrine (3 x 10-8 M). (
B
) Serotonin (10-6 M). See “Materials and Methods”
for details. Values shown are mean ± SE of four independent experiments.
*Significantly different from the control (
p
< 0.05).
8
6
4
2
0
0 5 10 25Arsenite (µM)
MLC-P/MLC
A
0
–++++
B
C
Arsenite (µM) 510 250
Arsenite (µM) 510 2500
Phenylephrine (10–8)–+++ +
MLC-P
MLC-P
MLC
Phenylephrine (10–8)
Figure 4. Effect of arsenite on MLC phosphorylation in aortic rings. (
A
) Basal levels of MLC-P. (
B
) MLC and
MLC-P levels stimulated by 10-8 M phenylephrine. See “Materials and Methods” for details. (
C
) Densitometric
analysis of MLC-P/MLC (typical results of one of three independent experiments).
by 10–50 mM K
+
in aortic rings without
endothelium, which is consistent with the
results shown in Figure 3. The next experi-
ment was performed using aortic strips
loaded with fura-2 fluorescent dye to investi-
gate whether arsenic increased intracellular
Ca
2+
in the presence of high K
+
. Arsenite,
however, did not induce intracellular Ca
2+
elevation, but rather decreased Ca
2+
concen-
tration significantly in the presence of
90 mM K
+
(Figure 5B). These results show
that arsenite enhances vasoconstriction with-
out concomitant increase of intracellular
Ca
2+
levels, suggesting that arsenite might
increase the contractility of blood vessels via
Ca
2+
sensitization.
To investigate this assumption, we meas-
ured smooth muscle contraction and the
change of intracellular Ca
2+
levels simultane-
ously using fura-2–loaded aortic strips. After
the muscle strips were treated with 25 µM
arsenite, contraction was induced successively
by 12.5, 25, and 90 mM K
+
. Figure 6A and
6B shows a scanned reduction of the tracings
from aortic strips. Compared with the control
group, arsenite-treated aortic strips showed
enhanced contraction without any concomi-
tant increase in intracellular Ca
2+
elevation
(Figure 6A,B). Plotting this result reveals that
the aortic strips treated with arsenite showed a
steeper slope in the intracellular Ca
2+
elevation
versus contraction relationship (Figure 6C).
These results suggest that arsenite might
potentiate vasoconstriction by enhancing the
Ca
2+
sensitivity of contractile machinery in
smooth muscle.
To verify the effects of arsenite on blood
vessels in vivo, we monitored changes in blood
pressure after intravenous infusion of phenyle-
phrine into conscious rats (Figure 7). An
intravenous bolus of arsenite had no effect on
basal blood pressure. When rats were treated
with arsenite 2 hr before phenylephrine infu-
sion, the hypertensive effect of phenylephrine
was significantly potentiated (23.0 ± 3.2 vs.
36.8 ± 3.6 mmHg, p= 0.029; Figure 7B,C).
These results suggest that arsenite could
induce the enhancement of agonist-induced
vasoconstriction in vivo and this confirms the
previous in vitro results (Figure 1).
Discussion
In the present study we demonstrate the abil-
ity of arsenic to enhance contraction of iso-
lated aortic rings from rats. We have shown
that arsenite enhances the vascular contrac-
tion induced by phenylephrine, serotonin,
and high K
+
in a concentration-dependent
manner and that the Ca
2+
sensitization in
smooth muscle largely contributes to arsen-
ite-induced hypercontractility. These in vitro
results were consistent with in vivo results in
which arsenite potentiated the hypertensive
effect of phenylephrine. Recently, the effect
of arsenic on platelets has been suggested as
a key mechanism in the development of
cardiovascular diseases (Lee et al. 2002).
Enhancement of vasoconstriction induced by arsenic
Environmental Health Perspectives
•
VOLUME 113 |NUMBER 10 | October 2005
1333
BA
10
4
3
2
1
0
Contraction (g)
K+ (mM)
Control
Arsenite
0.10
0.08
0.06
0.04
0.02
0.00
K+ (mM)
20 30 40 50 12.5 25.0 90.0
Control
Arsenite
∆ ratio (340/380 nm)
*
*
Figure 5. Effect of arsenite on vasoconstriction and intracellular Ca2+ levels in the presence of high K+.
(
A
) Contraction induced by various concentrations of K+in Ca2+-free KR solution in aortic rings without
endothelium after treatment with arsenite. (
B
) Intracellular Ca2+ levels determined in the presence of K+in
fura-2 loaded aortic strips without endothelium after treatment with arsenite; values shown are mean ± SE
of more than three independent experiments. See “Materials and Methods” for details.
*Significantly different from the control group (
p
< 0.05).
B
A
∆ Contraction (g)
Control
Arsenite
0.08
0.04
0.00
0.08
0.04
0.00
1.2
0.8
0.4
0.0
1.2
0.8
0.4
0.0
∆ Ratio (340/380 nm)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
∆ Contraction (g)
0.02 0.04 0.06 0.08
∆ Ratio (340/380 nm)
3 min
12.5 25 90
K+ (mM)
Control
Arsenite
Control
Arsenite
C
Figure 6. Simultaneous measurement of Ca2+ increase and contraction by KCl in aortic strips without
endothelium after treatment with arsenite. Representative tracings of intracellular Ca2+ increase (
A
) and
contraction (
B
) induced by KCl. See “Materials and Methods” for details. (
C
)Ca
2+ elevation versus the
magnitude of contraction; values shown are mean ± SE of seven independent experiments.
A
Blood pressure (mmHg)
140
120
100
80
–120
Time (min)
Saline
Arsenite
Arsenite
B
C
140
120
100
80
140
120
100
80
0123 456
Saline
Phenylephrine
Phenylephrine
Figure 7. Effect of intravenously administered arsen-
ite on phenylephrine-induced blood pressure
increase in rats. (
A
) Saline 2 hr after arsenite expo-
sure. (
B
) Phenylephrine 2 hr after saline exposure
(∆mmHg = 23.0 ± 3.2; mean ± SE of four animals).
(
C
) Phenylephrine 2 hr after arsenite exposure
(∆mmHg = 36.8 ± 3.6; mean ± SE of four animals).
See “Materials and Methods” for details. Periods of
infusion are indicated in each panel. Data are repre-
sentative tracings of four independent experiments.
Blood vessels, however, are another impor-
tant tissue in cardiovascular system. Hyper-
contractility of blood vessels disrupts
vasomotor tone regulation and makes vaso-
constriction predominate, which can induce
hypertension, complicating cardiovascular
disease. Elevated peripheral resistance has
been reported in populations exposed to
arsenic-contaminated drinking water and in
animals treated with arsenite (Carmignani
et al. 1985; Chen and Yen 1964). Our data
provide evidence that arsenite could enhance
vascular smooth muscle contractility, suggest-
ing that arsenic-induced hypercontraction of
blood vessels might be another mechanism
for arsenic-associated cardiovascular disease
observed in human populations.
Suppression of nitric oxide production in
endothelium results in the loss of vasodilator
activity, causing vasoconstriction (Moncada
et al. 1991), and we previously demonstrated
that arsenite disrupts endothelium-dependent
vasorelaxation via inhibition of endothelial
nitric oxide production (Lee et al. 2002). Pi
et al. (2003) also reported that vasoconstric-
tion was increased in aortic rings isolated from
arsenate-exposed rabbits. They explained the
phenomena as a result of impaired nitric
oxide formation. To examine whether the
hypercontraction of blood vessels observed
(Figure 1) was also mediated by the inhibi-
tion of endothelium-derived vasorelaxation
activity, we performed experiments in aortic
rings without endothelium. Surprisingly,
contraction by phenylephrine or serotonin
was still enhanced by arsenite treatment
(Figure 3), suggesting that the hyper-
contraction was dependent on smooth muscles
in blood vessels. Because arsenite not only
interferes with endothelium-dependent
vasorelaxation but also enhances smooth
muscle-dependent contraction, arsenite treat-
ment could result in overall hypercontraction
of blood vessels, leading to a possible increased
risk for development of vascular diseases such
as hypertension and atherosclerosis (Bohr et al.
1991; Mulvany and Aalkjaer 1990; Rubanyi
1993; Vanhoutte 1997).
As shown in Figure 2, treatment with arse-
nate (pentavalent inorganic arsenic) also
potentiated phenylephrine-induced contrac-
tion, whereas MMA
V
and DMA
V
showed no
significant effect. Arsenate is generally known
to exert its toxic effects by replacing phosphate
in various biochemical reactions because it has
a similar structure and properties to phosphate
(Oremland and Stolz 2003). However, it is
not certain that such properties are engaged in
the enhancement of vasoconstriction shown in
this study. Indeed, arsenate can be reduced to
arsenite at a comparable rate in cell systems,
and the biological effect of arsenate may in
part result from its reduction to arsenite
(Huang and Lee 1996). Therefore, consider-
ing that the effective concentration of arsenate
is higher than that of arsenite, it is possible
that the effect of arsenate arises from arsenite
formed by reduction of arsenate.
Phenylephrine and serotonin act on the
α
1
-adrenoceptor and the 5-HT
2
serotonin
receptor, respectively, on aortic smooth mus-
cle cells and thus result in increasing intracel-
lular Ca
2+
(Bruckner et al. 1984; Peroutka
1984). In contrast to these receptor agonists,
a high concentration of K
+
bypasses receptor-
signaling pathways and leads directly to intra-
cellular Ca
2+
elevation by opening Ca
2+
channels following membrane depolarization
(Chiu et al. 1987). These intracellular Ca
2+
increases by phenylephrine, serotonin, and
high K
+
result in phosphorylation of MLC
leading to vascular smooth muscle contrac-
tion. It is widely accepted that the degree of
MLC phosphorylation is the essential factor
that determines the extent to which smooth
muscle contracts (Fukata et al. 2001; Walsh
1994). As shown in Figure 4B and 4C,
MLC phosphorylation to phenylephrine was
augmented by arsenite, from which it can be
concluded that hypercontraction by arsenite
is an MLC phosphorylation-dependent
effect. In contrast, arsenite alone did not
induce the MLC phosphorylation significantly
(Figure 4A), consistent with the fact that basal
tones of blood vessels were not changed by
arsenite treatment alone.
In addition to intracellular Ca
2+
elevation,
the level of responsiveness of contractile
machinery to intracellular Ca
2+
plays a key
role in regulating the contractility of smooth
muscle cells (Somlyo and Somlyo 2000).
Simultaneous measurement of contraction
and intracellular Ca
2+
increase in aortic strips
revealed that arsenite potentiated the magni-
tude of contraction without concomitant
increase in Ca
2+
elevation (Figure 6), suggest-
ing significant contribution of Ca
2+
sensitiza-
tion to the hypercontraction induced by
arsenite. Previous studies suggested that the
mechanism for Ca
2+
sensitization was mainly
due to modulation of MLC phosphatase
activity (Fukata et al. 2001; Uehata et al.
1997). That is, if MLC phosphatase was
inhibited, the enhanced MLC phosphoryla-
tion could be elicited at the same Ca
2+
concen-
tration, which resulted in hypercontraction.
However, it is currently uncertain whether
arsenite could inhibit MLC phosphatase
directly.
In this study, we showed that arsenite
causes enhanced vasoconstriction in vitro
and demonstrated that Ca
2+
sensitization in
smooth muscle is responsible for MLC
phosphorylation-dependent hypercontraction
by arsenite (Figure 8). In our in vivo study,
arsenite treatment potentiated the hyper-
tensive effect of phenylephrine in rats. These
results confirm our in vitro observations and
suggest that increased vasocontraction may
be a contributing factor in the development
of cardiovascular diseases in populations
exposed to arsenic.
Lee et al.
1334
VOLUME 113 |NUMBER 10 | October 2005
•
Environmental Health Perspectives
SMC
Contraction
Arsenite
MLC
Ca2+
SR
Ca2+
Ca2+
Ca2+ –CaM
Ca2+ -sensitization
MLCK
PPase
MLC- P
IP3
Agonist High K+
Rc
GPLC
PIP2
Figure 8. Proposed mechanism for arsenite-induced vasoconstriction. Abbreviations: Ca2+–CaM, calcium
calmodulin; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol-4,5-bisphophate; PLC, phospho-
lipase C; PPase, phosphatase; SMC, smooth muscle cells; SR, sarcoplasmic reticulum. Arsenite enhances
the contraction of SMC by agonists such as phenylephrine or serotonin mediated through MLC phosphory-
lation. Arsenite also enhances vasoconstriction induced by high K+. The mechanism for this effect is due
not to alteration of intracellular Ca2+ levels, but to Ca2+-sensitization (shaded area) possibly via inhibition
of PPase.
Enhancement of vasoconstriction induced by arsenic
Environmental Health Perspectives
•
VOLUME 113 |NUMBER 10 | October 2005
1335
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